RoboSep™ - Stemcell TechnologiesThe sample tube containing the cells is placed in the specially...

RoboSep™The Fully Automated Cell Separator

T E C H N I C A L M A N U A LV E R S I O N 2 . 4 . 0

THIS PRODUCT IS MANUFACTURED AND CONTROLLED UNDER A QUALITY MANAGEMENT SYSTEM CERTIFIED TO ISO 13485 MEDICAL DEVICE STANDARDS.

FOR RESEARCH USE ONLY. NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES.

TOLL FREE PHONE 1 800 667 0322 PHONE +1 604 877 0713 VERSION 2.4.0

[email protected] [email protected] FOR GLOBAL CONTACT DETAILS VISIT WWW.STEMCELL.COM DOCUMENT #28940

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SAFETY INFORMATION AND WARNINGS

This instrument is intended for research use only and must be operated by trained professionals.

To ensure adequate cooling of RoboSep™, place it on a clean, flat surface, and be sure that there is at least 10 cm (4 in) of clearance

around each side of the instrument. Make sure that the bench space is free of any foreign objects or debris that could block the fan

vents.

Always connect the power supply to a 3-prong, grounded AC outlet rated 4A, 110–240 V using the AC power cord provided with

RoboSep™. Do not use an adapter to a two-terminal outlet. Before plugging the instrument in, be sure that the correct fuses are

installed. Use two Type F 250V 4A 5X20mm fuses.

Electrical devices pose the risk of electric shock. To reduce the risk of shock, do not open any covers that are fastened with screws.

While RoboSep™ is designed to withstand spills on its exterior surface, do not allow fluids to enter the interior of the instrument. In the

event of such a spill, disconnect the power cable before cleaning up.

Do not operate RoboSep™ in extreme humidity or in conditions that can create condensation. Protect the instrument against dust and

moisture, and avoid physical shock and strong forces. Do not exert excess pressure on the screen.

The EasySep™ magnets on the RoboSep™ carousel generate strong magnetic fields. Keep away from pacemakers, watches and

other objects that respond to magnetic fields.

As RoboSep™ weighs 32.6 kg (72 lb), exercise caution when moving the instrument. It is recommended that two people are present to

lift or move the instrument safely.

In case of malfunction, call STEMCELL Technologies Inc for service. Contact information is located at the back of this manual. Do not

attempt to fix the instrument yourself. Never remove the outer casing of RoboSep™. There are no user-serviceable parts inside the

instrument.

NOTICE

RoboSep™ is designed and certified to meet UL-CAN/CSA-CENELEC EN 61010-1 safety requirements. Safety certified products are

safe to use when operated in accordance with the technical manual. This instrument should not be modified in any way. Alteration of

this instrument will:

Void the manufacturer’s warranty.

Void the 61010-1 safety certification.

Create a potential safety hazard.

This product conforms to the “Class A” standards for electromagnetic emissions intended for laboratory equipment applications,

according to FCC (Part 15) and CENELEC EN 61326. It is possible that emission from this product may interfere with some sensitive

appliances when placed nearby or in the same circuit as these appliances. The user should be aware of this potential, and take

appropriate measures to avoid interference.

STEMCELL Technologies is not responsible for any injury or damage caused by the use of this instrument for purposes other than

those for which it is intended, or by modifications of the instrument or software not performed by STEMCELL Technologies or an

authorized agent.

Windows is a registered trademark of Microsoft Corporation in the United States and other countries.





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Table of Contents Safety Information and Warnings ........................................................................................................... ii

Notice ........................................................................................................................................................ ii

1 Introduction ......................................................................................................................................... 1 1.1 System Description .................................................................................................................................................................... 1

1.1.1 EasySep™ Magnetic Cell Separation Technology ............................................................................................................... 1 1.1.2 Robotic System Overview ..................................................................................................................................................... 2 1.1.3 The Robotic Arm and Carousel ............................................................................................................................................. 3 1.1.4 The Tip Stripping Arm ........................................................................................................................................................... 4 1.1.5 The Hydraulic System ........................................................................................................................................................... 4 1.1.6 Homing the Carousel and Robotic Arm ................................................................................................................................. 4

1.2 Instrument Set-Up ...................................................................................................................................................................... 5 1.2.1 Unpacking the Instrument ..................................................................................................................................................... 5 1.2.2 Instrument Placement ........................................................................................................................................................... 5 1.2.3 Power and Data Connections................................................................................................................................................ 5 1.2.4 Powering Up and Shutting Down .......................................................................................................................................... 5 1.2.5 Priming the System ............................................................................................................................................................... 6

1.3 Running RoboSep™: Quick Start Instructions ........................................................................................................................... 6

2 Running RoboSep™: Detailed Instructions ..................................................................................... 7 2.1 Introduction................................................................................................................................................................................. 7 2.2 Setting Up a Run ........................................................................................................................................................................ 7

2.2.1 Entering a User ID ................................................................................................................................................................. 7 2.2.2 Selecting Protocols ................................................................................................................................................................ 7 2.2.3 Preparing Cells ...................................................................................................................................................................... 8 2.2.4 Reagents and Resources ...................................................................................................................................................... 8 2.2.5 Loading the Carousel ............................................................................................................................................................ 9 2.2.6 Entering Reagent Lot ID with a Barcode Scanner .............................................................................................................. 10 2.2.7 Running the Separation ....................................................................................................................................................... 10 2.2.8 Collecting Cells .................................................................................................................................................................... 10 2.2.9 Removing the Carousel ....................................................................................................................................................... 11 2.2.10 Pause and Abort Functions ................................................................................................................................................. 11

2.3 Instrument Care ....................................................................................................................................................................... 12 2.3.1 Maintenance Tab ................................................................................................................................................................. 12 2.3.2 Aseptic Operation of RoboSep™ ........................................................................................................................................ 12

2.4 Report Files .............................................................................................................................................................................. 13 2.4.1 End of Run Report Files ...................................................................................................................................................... 13 2.4.2 Log Files .............................................................................................................................................................................. 13 2.4.3 Accessing Report Files ........................................................................................................................................................ 13

3 Customizing RoboSep™ .................................................................................................................. 14 3.1 Protocol Databases .................................................................................................................................................................. 14

3.1.1 The Global Database .......................................................................................................................................................... 14 3.1.2 Creating and Modifying Custom Databases ....................................................................................................................... 14 3.1.3 Loading Databases ............................................................................................................................................................. 14

3.2 Custom Protocols – RoboSep™ Protocol Editor ..................................................................................................................... 15 3.2.1 Start-Up Protocol Editor ...................................................................................................................................................... 15 3.2.2 Editing an Existing Protocol ................................................................................................................................................. 15





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3.2.3 Writing a New Protocol ........................................................................................................................................................ 16 3.3 Protocol Structure .................................................................................................................................................................... 17

3.3.1 Global Protocol Details ........................................................................................................................................................ 17 3.3.2 Protocol Command Sequence and Command-Specific Details ......................................................................................... 17 3.3.3 Advanced Protocol Editor Features .................................................................................................................................... 21

3.4 Saving a Protocol ..................................................................................................................................................................... 21 3.5 Printing a Protocol .................................................................................................................................................................... 21

4 Communications and File Transfer................................................................................................. 22 4.1 Set-Up for Network Connection ............................................................................................................................................... 22 4.2 Locating and Connecting to RoboSep™ on the Network ........................................................................................................ 22 4.3 Adding Protocols Remotely ...................................................................................................................................................... 22 4.4 Accessing Reports and Log Files Remotely ............................................................................................................................ 22 4.5 Transferring Protocols via USB Key ........................................................................................................................................ 22

5 Care and Maintenance ..................................................................................................................... 23 5.1 General Considerations ........................................................................................................................................................... 23 5.2 Daily Maintenance .................................................................................................................................................................... 23 5.3 Cleaning the Tip Head ............................................................................................................................................................. 23 5.4 Cleaning RoboSep™ ............................................................................................................................................................... 23 5.5 Tip Strip Failure Detection ....................................................................................................................................................... 24 5.6 Replacing the Fuses ................................................................................................................................................................ 24

6 Troubleshooting ............................................................................................................................... 25

7 Specifications ................................................................................................................................... 26 7.1 Factory Calibration ................................................................................................................................................................... 26 7.2 Security .................................................................................................................................................................................... 26

8 RoboSep™ Spare Parts and Accessories ...................................................................................... 27

9 Warranty ............................................................................................................................................ 27





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Pour off supernatant. Positive Selection: Desired, labeled cells remain in the tube. Negative Selection: Desired unlabeled cells are in the new tube.

Place tube in magnet

Incubate

Incubate

Add EasySep™

selection cocktail

Cell suspension

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Add EasySep™

magnetic particles

1 INTRODUCTION

RoboSep™ (Catalog #20000) is a laboratory instrument that automates EasySep™ immunomagnetic cell separation. EasySep™ is a

column-free magnetic cell separation technology that enables both positive and negative selection of desired cells from virtually any

species. Using an integrated color touch screen, RoboSep™ can be programmed to purify cells from up to four samples at once. The

user simply loads the consumables as indicated on the built-in touchscreen and starts the experiment; RoboSep™ then automatically

executes the EasySep™ immunomagnetic labeling and magnetic separation steps using disposable pipette tips.

RoboSep™ is intended for research applications only. Please make sure that you have read page ii of this manual before using the

instrument.

1.1 SYSTEM DESCRIPTION

1.1.1 EasySep™ Magnetic Cell Separation Technology EasySep™ is a powerful immunomagnetic cell selection procedure that

combines the specificity of monoclonal antibodies with the simplicity of a column-

free magnetic system. EasySep™ can be used as either a positive or a negative

selection approach to purify your cells of interest.

With EasySep™ positive selection, highly purified cells are obtained by

targeting the cells of interest with antibodies directed against one or more

specific cell surface antigens. These targeted cells are cross-linked to

EasySep™ magnetic particles. The sample tube containing the cells is then

placed directly in a specially designed EasySep™ magnet. The unique magnet

generates a high gradient magnetic field in the interior cavity that is strong

enough to separate cells labeled with EasySep™ magnetic particles without the

additional magnetic field gradients provided by a column matrix. The cells that

are not bound to the magnetic particles are removed either by pipetting off

(RoboSep™) or by inversion of the magnet (manual EasySep™). To obtain a

highly purified cell population, the cells remaining in the tube can undergo

additional separations prior to being collected by removing the tube from the

magnet (Figure 1). Unlike larger particles used with other column-free systems,

the EasySep™ magnetic particles used in positive selection procedures do not

interfere with subsequent flow cytometric analysis and do not need to be

removed.

For EasySep™ negative selection applications, unwanted cells are targeted for

depletion using a cocktail of monoclonal antibodies directed against specific cell

surface antigens. The labeled unwanted cells are then cross-linked to EasySep™

magnetic particles. The sample tube containing the cells is placed in the specially

designed EasySep™ magnet. The desired (unlabeled) cells are removed either

by pipetting off (RoboSep™) or by inversion of the magnet (manual EasySep™).

Figure 1- RoboSep™ fully automates the EasySep™ procedure for positive (illustrated below) or negative selection.





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1.1.2 Robotic System Overview RoboSep™ is a pipetting robot comprised of four main mechanical components that are operated under computer control (Figure 2):

The Robotic Arm

The Carousel

The Tip Stripping Arm (not visible in Figure 2)

The Hydraulic System

With these systems acting in concert, RoboSep™ performs the following actions to automate EasySep™:

Accurately dispenses magnetic labeling reagents into the cell samples using disposable 1 mL tips

Mixes and transfers the sample between tubes using a 5 mL tip

Transfers buffer from the buffer bottle to tubes on the carousel using a second 5 mL tip

Keeps track of the appropriate incubation times Scheduling software sets the sequence in which RoboSep™ will execute all of the actions

required to process up to 4 samples. This gives RoboSep™ the flexibility to perform different

types of separations at the same time. A custom-designed single-use tip rack contains all the

pipette tips necessary to process a single sample (Figure 3). Each tip has its own compartment

to prevent cross-contamination and the rack is sterilized by gamma-irradiation.

NOTE: Remove the lid of the tip rack prior to use.

Carousel

Robotic Arm

Lid

Buffer Bottle (may not be exactly as shown)

Touchscreen

Figure 2 - RoboSep™ Instrument

(a) With lid (b) Without lid

Figure 3 - RoboSep™ filter tip rack





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1.1.3 The Robotic Arm and Carousel The robotic arm (Figure 4) is driven by two independent stepper motors to provide rotation and vertical movement. Rotation of the

carousel is driven by a single stepper motor. The carousel is divided into 4 quadrants (Figure 5); a quadrant defines the minimum

functional area for processing a sample. Each quadrant is designed to accommodate a 14 mL sample tube, a magnet with a 14 mL

separation tube, up to 3 reagent vials, a tip rack and 2 multifunctional 50 mL tubes.

By coordinating the rotational and vertical movement of the arm

with the rotation of the carousel, RoboSep™ picks up disposable

tips from the custom tip racks on the carousel and then uses

them to transfer fluids from one container to another or to mix the

fluids in a given tube. Fluids are dispensed into the sample liquid

to ensure aerosol containment.

The robotic arm can pick up both 1 mL and 5 mL disposable

pipette tips using a unique tip head that has two conical tip

sealing surfaces (Figure 6). This dual tip capability enables

RoboSep™ to accurately dispense volumes from 12.5 µL up to

10 mL. RoboSep™ uses 1 mL tips to aspirate and dispense the

magnetic labeling reagents and 5 mL tips to transport the sample

and buffers.

Figure 5 – The carousel is divided into quadrants as pictured below. This figure shows each quadrant fully loaded with a magnet, tip rack, reagent vials, and tubes required to carry out a separation.

Figure 6 – The tip head (label A) has two conical surfaces that enable it to engage with the 1 mL (B) and 5 mL (C) disposable pipette tips.

Figure 4 – The robotic arm is capable of vertical and rotational motion. The stainless steel tip head at the end of the arm is used to pick up pipette tips.





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Flag

1.1.4 The Tip Stripping Arm RoboSep™ disposable tips are returned to the tip rack after each use. This eliminates the need for a waste tip area on the instrument

and facilitates easy clean-up after a run by allowing the entire tip box to be disposed of appropriately. Figure 7 illustrates the automated

pipette tip removal process.

1.1.5 The Hydraulic System The hydraulic system uses sterile deionized water driven by a positive displacement piston pump. The pipette handling tip head is

connected to the reservoir bottle containing the sterile deionized water via the pump. The only visible parts of this system are the water

bottle and the tubing running from the console to the robotic arm. The hydraulic fluid does not make contact with the user’s samples or

reagents, and because it is deionized water, it is less damaging to the piston seal than salt solutions or buffers. The RoboSep™ pump

therefore has a substantially longer operating life than the seals on syringe pumps used in other instruments and does not require any

user maintenance. The use of water as a hydraulic fluid instead of air allows for accurate high-speed dispensing: the water effectively

acts as an incompressible extension of the pump piston, allowing the system to operate with a dead air volume similar to that of a

manual pipettor.

1.1.6 Homing the Carousel and Robotic Arm RoboSep™ tracks the position of the robotic arm and carousel relative to physical markers called homing flags. A home position is

registered by the computer when a homing flag trips an optical sensor. The carousel homing flag is readily visible as a projection on the

bottom plate (Figure 8). This projection trips an optical sensor which can be seen on the front inside wall of the bowl when the carousel

is removed. The robotic arm homing flags and sensors are not visible. Homing of the carousel and robotic arm is performed

automatically at start-up, at the beginning of each cell separation experiment and after the instrument has been paused during a run.

The robotic arm home positions are detected again periodically throughout a run.

To ensure correct operation of the instrument, it is essential that no

mobile parts be moved or touched during an experiment. Homing is the

only mechanism by which RoboSep™ determines the location of items on

the carousel; any disruption of the carousel or the arm during operation can

cause pipetting actions to fail. In order to prevent such disruptions,

RoboSep™ operates with a transparent lid over the arm and carousel. If the

lid is opened during an experiment, the instrument will pause once the

current action is complete. Once the lid is closed, RoboSep™ will

automatically execute a homing sequence and resume the experiment.

Figure 7 – Disposable 5 mL pipette tip being replaced in the tip rack. The robotic arm places the used tip part way into the rack (A). The tip stripping arm swings out to engage the tip head just above the tip (B). The robotic arm then moves up and the tip falls into the tip rack (C).

Figure 8 – The carousel home flag is visible as a projection from the bottom plate in this schematic diagram





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1.2 INSTRUMENT SET-UP

1.2.1 Unpacking the Instrument A STEMCELL Technologies authorized agent will be present to help you unpack and install the instrument. Please note that proper

operation of RoboSep™ requires the correct installation of the instrument.

1.2.2 Instrument Placement To ensure adequate cooling of RoboSep™, the instrument should be placed on a clean flat surface with at least a 10 cm (4 in)

clearance around each side. Make sure that the bench space is free of any foreign objects or debris that could block the instrument’s

fan vents.

RoboSep™ is designed to fit in most biocontainment hoods for sterile operation; it is not specified for use in a cold room or refrigerator

(2 - 8°C, 39°F) (see Section 7 – Specifications). STEMCELL Technologies recommends operating RoboSep™ with the instrument’s lid

in place to prevent accidentally interfering with the operation of the robotic arm. Talk to your STEMCELL Technologies authorized

agent if you wish to remove the lid from RoboSep™ for installation in a biocontainment hood.

1.2.3 Power and Data Connections Plug in the power cord provided with RoboSep™ to the power supply

outlet located at the bottom right side of the instrument, next to the fuse

box and the power switch (Figure 9), and connect to a 3-prong, grounded

AC outlet rated 4A, 110–240 V. Do not use an adapter to a two-terminal

outlet.

Data connections to RoboSep™ are through an Ethernet port and 2 USB

ports. Section 4 – Communications and File Transfer provides more

detailed instructions on setting up RoboSep™ on a network.

1.2.4 Powering Up and Shutting Down Power up RoboSep™ by activating the power switch located at the

bottom right side of the instrument (Figure 9). RoboSep™ first loads the

computer operating system and then the instrument control software and user interface. The touchscreen provides visual confirmation

that these processes are occurring. After a short period of time, the robotic arm and carousel will move. The start-up sequence is

complete once the start screen is visible (see Figure 10 on page 7 for a screenshot).

Note: The start-up sequence may take longer than usual if a USB device is attached to RoboSep™.

The procedure to shut down RoboSep™ is as follows:

1. Navigate to the “Instrument Tasks” section of the graphical user interface by pressing the appropriate tab at the top of the touchscreen.

2. Under the “Maintenance” tab, press “Basic Shutdown Protocol.”

3. Wait until the screen becomes black. Once it has done so, turn off the instrument’s power switch.

Power cord

Switch

Figure 9 – Side view of RoboSep™ showing power and data connections, as well as the hydraulic fluid bottle and power switch.





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1.2.5 Priming the System RoboSep™ performs a priming sequence at the beginning of every cell separation protocol to minimize the effect of air bubbles that

naturally accumulate in the hydraulic system. However, when RoboSep™ is used for the first time, or if the hydraulic fluid bottle runs

empty, it is necessary to run a separate priming protocol by following the steps below:

1. Fill the hydraulic fluid bottle with 250 mL of sterile deionized water and connect the bottle to the hydraulic system tubing in the compartment located at the right of the instrument (Figure 9).

2. If the instrument is turned off, switch it on and wait until the end of the start-up sequence as described in section 1.2.4.

3. Press the “Instrument Tasks” tab, select “Basic Prime” under the “Maintenance” tab and then press the green “Load” button.

4. Place a 50 mL tube in the appropriate slot of the carousel as indicated on the screen.

5. Press “Run.”

If no water is expelled from the tip head into the waste tube by the end of the priming sequence, there could be a problem with the

connections at the hydraulic fluid bottle (see Section 6 – Troubleshooting).

1.3 RUNNING ROBOSEP™: QUICK START INSTRUCTIONS Note: Before using RoboSep™, ensure that the hydraulic fluid bottle is full (250 mL of sterile deionized water).

The following is a brief quick start guide to using the RoboSep™ fully automated cell separator; detailed instructions on running

RoboSep™ are provided in Section 2 of this manual. To quickly execute a run on RoboSep™:

1. Power up RoboSep™ and wait for initialization to complete.

2. Follow the instructions on the message bar at the top of the screen. Press “Select Protocol” to bring up the list of available protocols (see Section 3.1.2 to customize the protocol list). Choose the desired protocol from the list and then enter the sample volume. Repeat this process until all the desired protocols (up to four) are selected.

3. Press “Load” to begin the loading sequence. Lift the lid and load the indicated carousel quadrant(s) with the items listed on the left of the touchscreen. Press “Loaded” once each quadrant is ready. When all quadrants are loaded, close the lid.

4. Press “Run” and confirm you are ready for the instrument to start. RoboSep™ will now complete the selected protocols unless it is paused or halted. The time remaining until completion is displayed on the screen.

5. Once the run is complete, press “Unload,” open the lid, and collect the cells of interest. For positive selection, the cells of interest are in the magnet tube. For negative selection, the cells of interest are in the 14 mL sample tube of the second quadrant (if using 2 quadrant negative selection), or the 50 mL negative fraction tube of the first quadrant (if using 1 quadrant negative selection).

6. Unload the rest of the carousel and store the reagents appropriately. Discard the tip boxes containing the used tips in the appropriate waste container.

The instrument is now ready for another run. If no more runs are planned for the day, or if a spill occurred, clean the instrument as

detailed in Section 5 – Care and Maintenance. To conserve energy, turn off the instrument at the end of the work day.

Note: RoboSep™ protocols may differ from the standard manual separation protocols because they have been optimized for an

automated system. Unless otherwise noted, all RoboSep™ procedures have been optimized for use at room temperature (15 - 25°C),

regardless of the temperature recommended for the manual procedure.





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2 RUNNING ROBOSEP™: DETAILED INSTRUCTIONS

2.1 INTRODUCTION When the RoboSep™ cell separator is powered up (see power-up

instructions in section 1.2.4), the RoboSep™ graphical user interface

loads automatically; upon completion of start-up, the “Sample

Processing” start screen appears.

Note: Please wait for the end of the start-up sequence before touching

the touchscreen, the lid, or other moving parts of the instrument, so as

to not interfere with the start-up process.

The user interface consists of two main sections, defined by tabs at the

top of the screen: the “Sample Processing” section, which is shown at

start-up, and the “Instrument Tasks” section (Figure 10). The light grey

tab indicates which section is currently active. You can switch from one

section to the other at any time by touching the appropriate tab.

2.2 SETTING UP A RUN Navigate to the “Sample Processing” section of the user interface (Figure 10) by touching the appropriate tab at the top of the

RoboSep™ user interface. The sample processing screen is initially divided into five areas: Messages, Samples, Run Progress, User

ID, and a row of action buttons along the bottom. The areas’ functions are:

The Messages area indicates what step is next in the set-up process to start running RoboSep™.

The Samples area is divided into four rows corresponding to the four quadrants on the carousel. Each row has columns to display the protocol name, the sample volume (in mL), and the type of cell selection protocol. Once protocols have been selected, this area indicates on which quadrant of the carousel each protocol will be run.

The Run Progress area has a progress bar and countdown clock that shows the time to completion of the current run.

The User ID area allows the user to enter his/her unique code for reporting purposes.

The “Abort,” “Pause,” and “Load” action buttons allow the user to issue the appropriate commands to terminate, pause, or start a run.

2.2.1 Entering a User ID A User ID can be entered at any point before a run begins; it is stored internally and is included in the report file that is generated after

each run (see Section 2.4). User IDs are useful for keeping track of usage in a multi-user environment. To enter a User ID:

1. Press the grey box under the “User ID” heading. A numeric pad will pop up (note: a keyboard can also be used).

2. Key in the user ID, and press the “Enter” key.

2.2.2 Selecting Protocols RoboSep™ is delivered with a set of standard protocols optimized for use with “typical”

samples (e.g. peripheral blood mononuclear cells from normal donors). Standard

protocol names match the corresponding EasySep™ product name and catalog

number. Certain protocols also include a descriptor (e.g. “high recovery”) when more

than one protocol is provided for the same product. If working with atypical samples

(e.g. clinical/patient samples), protocols can be modified as appropriate and given a

different descriptor (see Section 3 – Customizing RoboSep).

The RoboSep™ protocol selection procedure is described below.

1. Choose a protocol to run: a. Touch “Select Protocol” in any row of the Samples area

(Figure 10). A list of the available protocols appears on the screen (Figure 11).

b. Select a protocol by touching the protocol name. A numeric pad pops up (Figure 12).

Figure 10 – Start screen of the RoboSep™ user interface

Figure 11 – List of protocols to select from





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c. Key in the sample volume in mL, and press the “Enter” key. The original “Sample Processing” screen will re-appear with the selected protocol in the appropriate quadrant row.

Note: A short description of the protocol appears in the Samples area.

2. Review/edit the information: a. To remove the protocol, press the “X” in the last

column.

b. To change the protocol, press the protocol name and repeat the protocol selection process above.

c. To change the sample volume while keeping the same protocol selected, press the sample volume for this protocol, and enter a new volume (Figure 12).

3. Repeat the protocol selection process until all the desired protocols are entered.

Up to four protocols can be chosen (they can be the same or different). When a negative selection protocol with two rounds of

separation is selected, the interface automatically assigns two adjacent quadrants for the protocol. Once three quadrants are occupied,

only protocols that use one quadrant will be displayed.

Protocol Order

The protocols are listed in order of last use, with the most recently used protocols at the top of the list (Figure 11). The protocols can

also be sorted by name or type. To change the basis of sorting, touch the appropriate column heading. For example, to sort protocols

in alphabetical order press the Protocol Name heading. An arrow in the heading indicates if the list is sorted in ascending or

descending order; touch the column heading again to toggle between these two options. The user can move up and down the list by

using the arrows at the top right corner of the protocols window.

2.2.3 Preparing Cells Refer to the Product Information Sheets that come with your EasySep™ reagents and/or magnet for instructions on how to prepare

cells for separation using RoboSep™. It is important to use the recommended cell concentration for best results. Ideally the sample

should be a single cell suspension. However, RoboSep™ tips will not clog if small aggregates are present.

2.2.4 Reagents and Resources The sample tube and separation tube are 14 mL (17 x 100 mm) round-bottom

tubes. We recommend Falcon™ 14 mL Polystyrene Round-Bottom Tubes (BD

Biosciences, Catalog #352057). The waste tube and negative fraction tube/lysis

buffer tube are 50 mL conical bottom tubes (e.g. BD Biosciences, Catalog

#352070). The Service Rack (Catalog #20101) is provided to hold the reagents

and sample prior to loading them on the carousel and to keep track of the caps

during a run (Figure 13). Like the carousel, the rack is laid out in quadrants, with

symbols on the reagent storage locations to assist with keeping tubes together

with the correct caps ( corresponds to the Magnetic Particles vial;

corresponds to Selection Cocktail vial; corresponds to Primary Antibody vial).

Figure 12 – Entering sample volumes

Figure 13 – The Service Rack is divided into quadrants and features symbols on the reagent storage locations to assist keeping tubes together with the correct caps.





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Minimum reagent volume required

Scroll button for reagent list

Waste Tube

Separation Tube

Negative Fraction / Lysis Buffer Tube

Sample Tube

Tip Rack

EasySep™ Reagent Vials

2.2.5 Loading the Carousel Once all the desired protocols have been selected and the cell samples have been prepared, the carousel can be loaded:

1. Press the “Load” button. If the same protocol has been selected in more than one quadrant, a pop-up window will open to give you the option of using a single set of reagents for all identical separations (Figure 14). Alternatively, one set of reagents may be used for each individual separation.

2. An image of the carousel will appear (Figure 15). The active quadrant number(s) is displayed at the bottom right of the window. At the top center of the window, the name of the protocol assigned to this quadrant is also displayed. On the left hand side of the window is a list of all the reagents and resources that need to be loaded for the current separation (see Section 2.2.4 for suitable tubes). The list starts with items near the center of the carousel and ends with items near the rim to facilitate aseptic loading. The user can move up and down the list by pressing the arrows located above and below.

3. Touch the first item on the list. The item will be highlighted in green and its location on the carousel indicated by a green circle. Turn the carousel so that the indicated quadrant is in a convenient position and place the item in the indicated location. Continue down the list, loading each item in turn.

4. Load reagents. The minimum reagent volume required for a given cell separation procedure is displayed below the name of each reagent. Verify that your vial has the minimum required volume. To assist in proper placement, the reagent vial labels have symbols that correspond to symbols on the carousel and on the touchscreen. For recording reagent lot numbers and sample IDs, first select the item on the list and then select the grey box under the “Reagent Lot ID” heading. Alternatively, STEMCELL offers a barcode reader (Catalog #20501) that can scan barcodes on reagent vials and sample tubes to record reagent lot # and sample IDs (see Section 2.2.6).

Important Notes:

RoboSep™ uses EasySep™ reagents in 1.5 mL vials from Sarstedt. Use of any other vial will cause RoboSep™

separations to fail.

Certain negative selection protocols use D magnetic particles that can be pulled to the side of the sample tube by

the external magnetic field of “The Big Easy” EasySep™ Magnet. While this does not affect performance, it can be

prevented by inserting a magnet shield between the magnet and the sample tube.

Figure 14 – One set of reagents can be used for multiple identical separations

Figure 15 – Carousel layout. The locations of the items listed on the left side of the screen are indicated with a green circle when selected. In this example, “Separation Tube” is highlighted in green on the left, and the location of the tube is indicated with a green circle in Quadrant 1.





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When loading the tip rack, make sure that the cap has been removed (see Figure 4). Be careful not to open the

lower joint when removing the cap from the tip rack. The rack must be fully seated so that the tips are in the correct

location for pick-up by the robotic arm.

The cap must be removed from the RoboSep™ Buffer Bottle (Catalog #20104) and ensure there is sufficient

volume, as indicated on the screen.

5. Press the green “Loaded” button when all the reagents and resources are loaded on the active quadrant(s). The reagents and resources required for the next selected protocol will appear.

6. Repeat steps 2 – 4 until all the required quadrants are loaded. Close the instrument lid. The instrument is now ready to run a separation.

2.2.6 Entering Reagent Lot ID with a Barcode Scanner Lot numbers on RoboSep™ reagents can be scanned and automatically recognized by a barcode scanner (Catalog #20501). The scanned information will record the lot number in the “Reagent Lot ID” field shown on the right hand corner of Figure 15. All recorded information can be retrieved from the RoboSep™ Log Files (see Section 2.4 Report Files) for convenient tracking of RoboSep™ reagents.

To use the barcode scanner, connect the scanner to the RoboSep™ unit via the USB outlet. Wait for a few seconds for the scanner to connect to the unit. A red scanning laser should appear when the scanner is triggered. Upon loading the carousel and recording the lot number, align the scanner and scan the barcode on the RoboSep™ reagent vial to record the lot number. The lot number should appear on the “Reagent Lot ID” field.

2.2.7 Running the Separation After the instrument has been readied for separation, the following steps will

start the experiment:

1. Press the green “Run” button (Figure 15) to start the cell separation experiment. A warning message will appear asking the user to verify that all the reagents and resources are correctly loaded and that nothing can interfere with the movements of the instrument. Please ensure that the lids have been removed from all vials and resources, including the buffer bottle.

2. Once everything is verified to be clear, press “Confirm” (Figure 16). Observe that the initial prime step ejects water from the tip head into the waste tube in carousel Quadrant 1 (Q1). This ensures that the hydraulic fluid system is properly connected.

The experiment is now in progress. The time remaining until completion is

displayed at the bottom of the screen (Figure 17). The machine will beep

when all separations are complete.

2.2.8 Collecting Cells To collect the cells of interest, press “Unload” and allow 5 seconds for the

robotic arm and carousel to move to a safe position before opening the

instrument lid. The position of the isolated target cells will be indicated on the

RoboSep™ touchscreen (Figure 18).

For positive selection:

Remove the 14 mL tube containing the isolated cells from the magnet. Promptly re-suspend the selected cells in an appropriate volume of buffer.

If desired, collect the 50 mL tube containing the depleted fraction located to the left of the tip box.

Figure 17 – Run Progress. Once a run has begun, progress is shown at the bottom left, with an indication of how much time remains.

Figure 16 – Warning message

Figure 18 – Following separation, the location of the desired cells is indicated by a green circle.





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For negative selection:

If two rounds of separation were performed, collect the enriched cells in the 14 mL tube located to the left of the magnet on the second quadrant used for the negative selection procedure.

If only one round of separation was performed, collect the 50 mL tube containing the enriched cells located to the left of the tip box.

If desired, collect the 14 mL tube(s) containing the labeled cells located in the magnet(s).

Once the separated cells have been collected: Unload the carousel and store the reagents appropriately.

Discard the tip boxes containing the used tips in the appropriate waste container.

If prompted, refill the hydraulic fluid bottle with sterile deionized water for further experiments.

The instrument is now ready for another experiment.

2.2.9 Removing the Carousel The carousel can be easily removed from the instrument for cleaning or to facilitate loading and unloading of the samples and

reagents. Some users may prefer to remove the magnets prior to removing the carousel to reduce its weight. Position the carousel with

Quadrant 1 on the front side of the machine (facing the user) before removing it so that the optical sensor is not damaged.

To remove the carousel:

1. Rotate the carousel so that Quadrant 1 is on the front side of the machine.

2. Unscrew the black central knob (Figure 19) by rotating counterclockwise until the threads have disengaged.

3. Lift out the carousel.

To replace the carousel within the instrument:

1. Rotate carousel so that Quadrant 1 is on the front side of the machine.

2. Place the carousel over the cylindrical bearing hub.

3. Align the holes around the knob with the pins on the bearing hub by slowly rotating the carousel.

4. Screw the central handle clockwise until tight. It is important to tighten the knob fully so that the carousel stays level when the robotic arm pushes down on it to pick up pipette tips.

2.2.10 Pause and Abort Functions Hitting the “Pause” button will cause RoboSep™ to interrupt the run as soon as it completes the current action. Pressing the “Resume”

button will restart the run at the next scheduled action after RoboSep™ orients itself by performing homing actions. The “Abort” button

also stops execution of the run as soon as the current action is complete, but with the option to exit from the protocol completely or to

resume the run.

Figure 19 – Close-up of knob used to fasten the carousel to the bearing hub





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2.3 INSTRUMENT CARE If no more experiments are to be performed that day, or if there was a spill on the

instrument, clean the instrument as detailed in Section 5 – Care and Maintenance.

2.3.1 Maintenance Tab To conserve energy, turn off the instrument at the end of the work day.

Navigate to the “Instrument Tasks” section of the graphical user interface (Figure 20).

Select “Basic Shutdown Protocol” from the “Maintenance” tab.

Wait until the screen becomes black, and then turn the switch on the right side of the instrument to the off position.

Two other protocols can be run from this page: “Basic prime” and “Home all.” If an

issue arises, STEMCELL Technologies Technical Support may ask that these

protocols be run and the results observed in order to assist with troubleshooting.

Note: The Service software and Package for Diagnosis can be launched from this window, allowing for calibration adjustment and diagnostics. This is not necessary unless requested by a STEMCELL Technologies representative.

2.3.2 Aseptic Operation of RoboSep™ To ensure aseptic operation, RoboSep™ must be used in a biocontainment hood. RoboSep™ is designed to fit easily in most

commercially available hoods. In addition, RoboSep™ does not affect sterility within the biocontainment hood; this has been verified by

particle count as per ISO standard 14644-1.

Although the hydraulic fluid is internal to the instrument and does not come into contact with the sample, reagents, or tips during

normal operation, the use of sterilized deionized water is recommended for aseptic operation.

Standard RoboSep™ protocols dispense fluids into the sample liquid to ensure aerosol containment. Aerosol-resistant filter tips are

supplied with each RoboSep™ kit.

Figure 20 – The shutdown protocol, located under Maintenance on the Instrument Tasks tab





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2.4 REPORT FILES Two types of usage data are automatically saved during RoboSep™ operation: Run Reports and Log Files. Run Reports contain

details of each run as entered by the operator, while Log Files contain more detailed information about instrument operation.

2.4.1 End of Run Report Files

Information such as the operator ID, protocol name, run time, reagent lot

numbers and usage volume, sample ID and volume is presented in an End of

Run Report. To benefit fully from the End of Run Report, the lot number, sample

ID and user ID information should be entered during experiment set-up (see

Section 2.2.5).

2.4.2 Log Files Log files are used by authorized STEMCELL Technologies personnel to assist

with troubleshooting and diagnostics.

2.4.3 Accessing Report Files The “Run Log” page, under the Instrument Tasks tab, lists diagnostic and run

information. The End of Run Report and Log Files are accessed via the buttons at the bottom of the page (Figure 21). Selecting the

green “Reports” button brings up a folder containing all of the reports that have been generated to date. These are organized into

subfolders by year and month. The Report file name is in the format “Report_DDMMMYYYY_HH_MM_SS.html”. The folder contains all

the run reports in chronological order.

Selecting the “Logs” button brings up a folder containing all of the log files created for your instrument. One file is created each time

RoboSep™ is initialized. The file names have the format robosep.log.XXX, where “XXX” is “txt” for the most recent file and “XXX” =

1, 2, 3… for increasingly older files.

To access the Report and Log files remotely, set up a connection between RoboSep™ and an external computer as explained in

Section 4 – Communications and File Transfer.

Figure 21 – The Run Log page, shown below, is used to access reports and log files.





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3 CUSTOMIZING ROBOSEP™

RoboSep™ is provided with a set of protocols optimized for use with “typical” samples. Using the provided RoboSep™ Protocol Editor,

users can customize protocols for their specific applications and add them to the global protocol database. Users can also customize

the list of protocols that are available to them during an experiment.

Please note: A keyboard and mouse should be connected to RoboSep™ before using the features described in this section.

3.1 PROTOCOL DATABASES Protocol databases are listed on the Profile page under the Instrument Tasks

tab (Figure 22). The Preset Protocol Databases list contains the global

protocol list (All) as well as lists based on desired cell species (All Human, All

Mouse, All Other) and sample type (e.g. All Whole Blood). The Custom

Users list can contain up to 5 user-defined protocol databases.

3.1.1 The Global Database A list of all the available protocols will appear (Figure 23) by selecting the

“Protocol List” button. Custom protocols can be added to the list by pressing

the “Add” button located at the bottom left of the screen from the Profile

page. This will bring up a standard Windows dialog box giving access to

protocol files on a USB memory stick or a shared folder on a networked

computer. See Section 3.2 for details on creating custom protocols.

To remove protocols from the global database, highlight a protocol and

select the “Remove” button. The selected protocol is moved from the

protocol folder to an archived sub-folder. To retrieve an archived protocol,

open the protocol with the protocol editor as described in Section 3.2.2 –

Editing an Existing Protocol – and save it in the main protocols directory.

3.1.2 Creating and Modifying Custom Databases RoboSep™ allows you to create up to 5 custom user protocol databases.

1. Select the “Modify/Update” button under the “Profile” tab. A new screen will appear (Figure 22).

2. Enter the desired User Name. This name will appear in the Custom Users list on the Profile page (Figure 22).

3. Select a protocol from the Current List of Protocols on the left and press the “Add” or “Remove” button, as appropriate (Figure 24). Repeat until the User Protocol list on the right contains all desired protocols.

4. Press “Save.” The User Name is now associated with the list of User Protocols

3.1.3 Loading Databases Go to the Profile page under the “Instrument Tasks” tab.

1. Select a Custom User or Preset Protocol Database (Figure 22).

2. Press “Load.”

The protocols in that database will now be available for use through the

“Sample Processing” tab.

Figure 22 – The profile page displays current custom user profiles and preset protocol databases.

Figure 23 – List of all currently available protocols

Figure 24 – Customization of protocols available to a specific user





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3.2 CUSTOM PROTOCOLS – ROBOSEP™ PROTOCOL EDITOR The RoboSep™ Protocol Editor is a stand-alone program that is installed on RoboSep™. The editor is used to modify existing

protocols or to write new ones.

Note: This program is not required for normal RoboSep™ operation.

3.2.1 Start-Up Protocol Editor To start the protocol editor, press the “Protocol Editor” button under the “Profile” tab (Figure 22). When the Protocol Editor is initially

opened, all the fields are grayed-out. To begin, either “New” or “Open” must be selected from the “File” drop-down menu. Select “New”

to begin creating a new protocol or “Open” to edit an existing protocol.

3.2.2 Editing an Existing Protocol Please contact STEMCELL Technologies Technical Support for assistance if required.

To edit an existing protocol with the Protocol Editor, click on “Open” on the “File” menu and select the desired protocol. This will

populate the editor screen with the commands and settings for that protocol. Once the desired protocol is opened, any of its settings

and fields can be changed (Figure 25). A description of the commands is found in Section 3.3 – Protocol Structure. When satisfied with

the edited protocol, click on “Check Protocol” to ensure that the new values and settings are correct. A screen will pop up if there are

errors in the Sample Volume and Working Volume values. Check the volumes highlighted in red on the pop up screen and click “OK” to

exit and modify the values on the Protocol Editor. Click on “Check Protocol” again and if the popup screen does not appear, access the

“File” menu from the top tool bar and follow the procedures under “Saving a Protocol” (Section 3.4) to save your work.

Notes:

To avoid over-writing the original protocol, the “Save As” option must be selected instead of “Save.”

The “About” menu will bring up details of the selected protocol and allow the name of the Author to be entered. The Author is for tracking purposes only and does not affect who can use the protocol.

The Label should include a Protocol Number in the name. Generally, this is the 5-digit number corresponding to the STEMCELL Technologies catalog number for the product used with the protocol. For more information, see Section 3.3.1.





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1. Protocol Type: Select the desired separation type from the

pull-down menu.

2. Label: Enter the name of the protocol as you wish it to

appear in the “Protocol Selection” screen on RoboSep™ (see

Figure 11); include a protocol number in the name—see Notes

above.

3. Description: A short description of the protocol will appear

in the Sample Volume dialog box just prior to loading the

protocol for a run (see Figure 12).

4. Sample Volume: Enter the minimum and maximum sample

volume permitted (see Section 3.3.1 for more information)

5. Working Volume Threshold: Enter the sample threshold

volume. RoboSep™ will use the “High” volume for top-up

commands with any sample above or equal to the threshold

volume. The “Low” volume will be used for top-up commands

with any sample smaller than the threshold volumes (see

Section 3.3.1).

6. Command Sequence: The list of programmed commands

will appear here in the order of execution.

7. Move Up & Move Down: Selected commands can be

moved up or down to change the order in which they will be

executed.

8. Add: Use the pull-down menu to select the type of

command to be added and click the “Add” button.

9. Command Details: Parameters that are specific to different

commands can be changed here.

Figure 26 – A list of preset standard separation templates

3.2.3 Writing a New Protocol Please contact STEMCELL Technologies Technical Support for assistance if you are using this for the first time.

In the “File” menu, click on “New.” A list of preset standard separation templates will appear (Figure 26). Select the desired separation

type from the list and click “OK”. Once the standard separation template is opened, any of its settings can be changed, commands can

be added or deleted, and the order of command execution can be changed. A description of the commands is found in Section 3.3 –

Protocol Structure. When satisfied with the protocol, access the “File” menu from the top tool bar and follow the procedure under

“Saving a Protocol” (Section 3.4) to save your work.

Figure 25 – Protocol Editor screen fields





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Figure 27 – Protocol type pull-down list

3.3 PROTOCOL STRUCTURE A protocol is defined by global protocol details, such as positive or negative selection, and by a list of commands with command-

specific details, such as incubation time. The global parameters are shown above the first red line in Figure 25, the command

sequence is shown below the first red line, and the command-specific parameters are shown below the second red line.

3.3.1 Global Protocol Details Note: See Figure 25 and Figure 26 for illustration of each point below.

Protocol Type: Select the appropriate protocol type from the pull-down list (Figure 27). The

protocol will be listed in the corresponding Preset Protocol Database. Maintenance and

Shutdown protocols are for service and diagnostic routines and are not useful for cell

separation.

Label: The name entered here is what will appear in the protocol list. This name must be

unique for the protocol to be registered as valid. The label should include a Protocol Number

in the name. This is the 5-digit number corresponding to the STEMCELL Technologies

catalog number for the product used with the protocol. Only letters, numbers, and dashes ()

should be used in the label name for proper processing by the RoboSep™ software.

Sample Volume Minimum and Maximum: These fields define the allowable range of

sample volumes. Minimum should be set such that all “relative volume” dispensing actions

are of at least 12.5 µL. For example: if particles are added at 50 µL/mL of cells, then the

minimum sample volume should be 250 µL (12.5 µL x 1000 µL/mL÷ 50 µL/mL= 250 µL).

Maximum should be set such that the total sample volume after all reagent additions is less

than 10 mL. For example: if cocktail is added at 100 µL/mL and particles at 50 µL/mL of cells, then the maximum sample volume

should be set at 8.5 mL (8.5 mL + (0.1 x 8.5) + (0.05 x 8.5) = 9.8 mL).

Working Volume Threshold (Low and High): After the magnetic labeling steps, RoboSep™ tops up the sample to a given working

volume before transferring the sample to the magnet for separation. The top-up volume can be set such that sample volumes less than

a given “Working Volume Threshold” are topped-up to a “Low” working volume, while sample volumes greater than or equal to the

threshold are topped up to the “High” working volume. This capability exists to improve separation performance for small volume

samples.

Suggested settings for negative selection protocols:

“Working Volume Threshold” = 4000 µL, “Low”= 5000 µL, “High” = 10000 µL.

Suggested settings for positive selection protocols:

“Working Volume Threshold” = between 1500 and 2000 µL, “Low”= 2500 µL, “High” = 10000 µL.

3.3.2 Protocol Command Sequence and Command-Specific Details A defined set of commands is used to automate cell separation processes on RoboSep™. These commands are Transport, Mix,

Incubate, Top-up/Resuspend, Pause, and Separate. To add a command:

1. Select a command from the pull-down menu in the “Command Sequence” section and click “Add” (Figure 25).

2. Enter the appropriate details (see below).

3. Click on the “Confirm Command” button in the “Command Details” section to confirm the settings.

4. Move command to desired position in command sequence using the “Move Up” and “Move Down” buttons.

The Command Sequence area can be enlarged by using the “Hide Header” button in the top right corner (Figure 25). Each command

has specific parameters that influence how it is executed. These will appear in the “Command Details” section of the editor console.

Label: The description in this box will appear in the “Command Sequence” list once all the details of the command have been entered.

Extension Time: Each command takes a set amount of time to execute. The extension time specifies what additional time is available

to RoboSep™ to facilitate the scheduling of all the required actions. The default extension times can be changed through the “Options”

pull-down menu. See Section 3.3.3 – Advanced Protocol Editor Features for further details.





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Source: This is the container (tube, reagent vial, or buffer bottle) from which the pipette will aspirate. If a reagent vial is specified as the

source, a 1 mL pipette tip is used automatically. If a sample, separation, or -negative fraction tube is the source, then the 5 mL tip

reserved for cell handling is used. If the source is the buffer bottle then the 5 mL tip reserved for buffer handling is used. The source is

chosen from a pull-down list which is sorted by relative quadrant number. The source tube should be in Quadrant 1 in most cases,

since Quadrant 1 is the start quadrant for all protocols. Tubes in Quadrant 2 are only accessed in protocols requiring two quadrants

(e.g. some negative selection protocols).

Destination: This is the container into which the pipette will dispense. The destination container is also selected via pull-down menu.

The list is sorted by relative quadrant number. A protocol with a source in Quadrant 1 and a destination in Quadrant 2 will occupy two

quadrants on the carousel. If this protocol is loaded in Quadrant 3, the destination will be in Quadrant 4.

Free-Air Dispense: When this check box is enabled, RoboSep™ dispenses the fluid that it is transporting just below the upper lip of

the destination container and above the liquid in the container. This option is useful where contact with the contents of the destination

tube is undesirable (e.g. preventing contamination of the designated buffer tip when adding buffer to the sample tube.).

Note: This option is not recommended for transfer of small volumes (e.g. addition of reagents).

Volume: For Transport commands, the user can specify whether RoboSep™ is to transport an absolute volume (in µL) or a volume

relative to the initial sample volume (e.g. to have RoboSep™ add 100 µL of reagent per mL of sample volume, program the addition of

a relative volume with a proportion of 0.10).

Incubation Time: This parameter specifies the length of time, in seconds, for reagent and magnetic separation incubations.





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Transport: This command is used to transport fluids from one vial or

tube to another (Figure 28). A label is required to describe the steps. The

source and destinations need to be appropriately specified, as well as

whether a volume relative to the sample volume or an absolute volume is

added (see “Volume,” above). The default tip rack is in Quadrant 1, but a

tip rack in another quadrant can be specified by activating the Tip Rack

check box. Free-Air Dispense can also be selected. For negative

selection protocols using D magnetic particles or Streptavidin

RapidSpheres™, the free-air dispense should be selected when

transporting from the sample tube to the selection tube. For all other

transports from the sample tube to the selection tube, the free-air

dispense should not be selected.

Mix: This command is used to mix the contents of a vial or tube. A label

is required to describe the step (Figure 29). The source is the tube

containing the sample or reagent to be mixed, and there is an option to

specify the extension time (see Section 3.3.3) and the tip rack to be

used. There are tip-use rules which decide which tip will be used for

each mixing step; these rules are automatically applied by RoboSep™.

Selecting “Home” prompts RoboSep™ to perform the “Home All”

command (see below) prior to the “Mix” step; this option is not necessary

for most RoboSep™ applications. If Relative Proportion is selected, a

fraction of the calculated volume in the vial will be mixed. Most standard

RoboSep™ mix steps use a relative volume of 0.33. If Absolute Volume

is selected, the set volume (entered by the user) will be mixed. The gap

between the mixing tip and the bottom of the tube (typically 100 µL)

should also be entered. The number of mix cycles can be modified from

1 to 5; most standard RoboSep™ mix steps iterate 3 times.

Incubate: This command allows for incubation of the sample with

reagents prior to another step in the sequence being performed

(Figure 30). A label is required to describe the step. As before, there are

options for extension time and home.

Separate: This command is functionally the same as the “Incubate”

command and is used to set incubation times for magnetic separations

(Figure 30). The separation time is the minimum amount of time before

the next command in the sequence is executed. Typical values for

separation time are between 150 and 900 seconds (2.5 and 15 minutes).

Figure 30 – Incubate or separate command

Figure 28 – Transport command

Figure 29 – Mix command





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Top-Up and Resuspend: “Top-Up” is used to dilute the sample with

RoboSep™ buffer before transfer to the magnet for separation (Figure 31).

The source is always the buffer bottle. The default top-up volume is specified

by the Working Volume Threshold, Low and High values (Figure 25). The top-

up volume can also be proportional to the sample volume (e.g. to add 2.0 mL

buffer per 1.0 mL sample, enter a relative proportion of 2). This command has

the same function as the “Resuspend” command. However, while “Top-Up” is

used to bring the sample to the desired working volume at the beginning of

the protocol, “Resuspend” is generally used to resuspend cells after magnetic

separation steps. Both commands should use a free-air dispense to prevent

contamination of the buffer tip with sample.

Home All: This command re-initializes the carousel and robot arm

movements (Figure 32). As there is an automatic re-initialization each time

RoboSep™ is turned on, or when a protocol is run, this option is not required

in standard separation protocols. A label is required and an extension time

should be entered to allow the protocol to continue at the end of the “Home

All” step (see following section). Alternatively, the components may be left in

their homed position (check box).

Pause: The pause command allows the user to intervene at a specific point

in a protocol (Figure 33). For example, the pause command could be used if

an application requires a centrifugal wash step at some point during

processing. The machine will beep and the Label text will appear on the

screen when the pause step is reached. Press the “Resume” button when

ready to continue.

Flush and Prime: These are used internally to prepare the hydraulic fluid

system for use. These commands are not needed in cell separation

protocols.

Figure 31 – Top-Up command

Figure 32 – Home All command

Figure 33 – Pause command





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3.3.3 Advanced Protocol Editor Features Additional capabilities can be accessed through the “Options” pull-down

menu (Figure 34).

By enabling “Extension Time” the user has access to the

extension time parameter for all commands. The default

extension times have been selected to give maximum

flexibility to the built-in protocol scheduler without impacting

separation performance. If the default times are altered, for

example to allow scheduling of unusual protocols, the desired

combination of protocols should be tested in a “wet” run (with

water) prior to use in a cell separation experiment to verify

that all actions are completed as expected.

The “Customize Vial Names” menu selection brings up a

dialog box that allows the user to change the names of tubes

and reagent vials as they appear on the Loading screen. This

feature can be used to create names in different languages,

or to make the names more appropriate for a given set of

custom applications. Highlight the default name you wish to

change and enter a new name (Figure 35).

Note: Names longer than 50 characters may not fit properly

on the user interface.

The “Multiple Samples Selection” menu allows the advanced

user to create customized protocols to process multiple

samples. Please contact STEMCELL Technologies for more

information.

3.4 SAVING A PROTOCOL The “Save” option will not be available until the protocol is validated by

the editor. To do this, click the “Check Protocol” button located at the

top-right corner of the editor console (Figure 25). If successful, the

symbol beside the “Check Protocol” button will turn from a cross (X) to

a tick () (Figure 36). The “Check Protocol” function ensures that all

information that is critical for protocol execution is present. It does not

ensure that your protocol is correctly programmed.

3.5 PRINTING A PROTOCOL Under the “File” menu, there is an option to print out the protocol. This

can be used for record keeping, debugging, or troubleshooting with

STEMCELL Technologies Technical Support.

B

A

Figure 34 – Options menu

Figure 35 – Customizing vial names

Figure 36 – The Check Protocol dialog (A) before and (B) after a successful protocol check. The file cannot be saved until the check is successful.





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4 COMMUNICATIONS AND FILE TRANSFER

Since RoboSep™ runs on Windows XP, it offers many possibilities for communication with other computers. RoboSep™ comes

configured for communication directly with another computer via crossover cable (supplied); RoboSep™ can easily be configured to be

connected to your network as well.

4.1 SET-UP FOR NETWORK CONNECTION When a STEMCELL representative installs your instrument, RoboSep™ can be set up to automatically obtain an IP address from the

local network. This will allow access to shared folders on RoboSep™ from any other computer on the network. If needed, consult the

local network administrator to verify that this is allowed and that the correct permissions are available.

4.2 LOCATING AND CONNECTING TO ROBOSEP™ ON THE NETWORK Whether RoboSep™ is connected to a network or directly to another computer, it must be located before its shared folders can be

accessed by other machines. If using Windows XP, follow these steps to locate the device and view RoboSep™ folders remotely:

1. Select “Search” from the Start Menu.

2. Select “Printers, computers, or people” then select “Computer on network.”

3. Enter RoboSep™ name Robo-XXXX where XXXX is the instrument serial number listed on the side of the instrument or in the “About” tab of the user interface.

4. Double click on the Robo-XXXX icon. You should now see the shared folders on RoboSep™. The shared folders are Protocols, Reports, and Logs.

5. Click on the desired folder and log on as “guest” when prompted.

For instructions on how to mount remote folders in other operating systems, contact the local network administrator.

4.3 ADDING PROTOCOLS REMOTELY RoboSep™ comes with pre-installed protocols that have been optimized for cell separation from normal samples using supported

EasySep™ products. Protocols that have been created for other applications using the protocol editor (see Section 3) can be copied

into the Protocols folder on RoboSep™. To do this, navigate to the RoboSep™ folders on the network (as shown in the previous

section) and then perform the following:

1. Copy the RoboSep™ protocol(s) from the source computer onto RoboSep™ by either “copying and pasting” or by simply “dragging and dropping” the protocol file into the Protocol Folder.

Note: The file being copied must have a unique name to avoid potentially overwriting existing protocols. Also, to secure the protocol against accidental modification, change the file properties to “read-only”.

2. Once the desired protocols have been transferred to RoboSep™, update the protocol database list(s) as described in Section 3.1.1.

3. Verify that the protocol behaves as expected with at least one dry run.

Protocols that are no longer required can be removed from the protocol folder and stored in an “Archive” sub-folder on RoboSep™ or

on any other networked computer.

4.4 ACCESSING REPORTS AND LOG FILES REMOTELY For complete information regarding RoboSep™ usage reports and log files, including file structure and naming conventions, please

refer to Section 2.4 on page 13. To access these files remotely, locate RoboSep™ on the local network (as described in Section 4.2);

the Reports and Logs are found under the appropriately-named folders.

4.5 TRANSFERRING PROTOCOLS VIA USB KEY 1. Insert your USB key into the USB port on your PC. Your PC will detect the USB key as a drive/storage device.

2. Transfer RoboSep™ protocol(s) to or from the USB key by “copying and pasting” or by simply “dragging and dropping”. Remove the USB key from the USB port when the transfer is complete.

3. Insert the USB key into the USB port on RoboSep™. Repeat step 2.





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5 CARE AND MAINTENANCE

5.1 GENERAL CONSIDERATIONS RoboSep™ is designed to avoid any cross-contamination of the samples and reagents manipulated during experiments. In addition,

RoboSep™ can be operated within a biocontainment hood to maintain sample and reagent sterility. It is the responsibility of the

operator to maintain a clean and uncluttered environment to ensure that RoboSep™ performs as expected.

When using RoboSep™, as during any research experiment, it is essential to observe good laboratory practices as described by your

institution or company.

5.2 DAILY MAINTENANCE The following should be performed daily to ensure longevity and reliability of RoboSep™:

1. Wipe down all exposed surfaces with an alcohol swab.

2. Verify that the hydraulic fluid bottle is full of sterile deionized water.

5.3 CLEANING THE TIP HEAD At regular intervals, a pop-up screen will prompt you to clean the RoboSep™ tip head. Please use the RoboSep™ Tip Head Polishing

Compound (Catalog #20119) provided in the RoboSep™ Accessory Kit to clean the RoboSep™ tip head – do not use ethanol or

isopropanol on the tip head. The tip head can accumulate a thin film of plastic from repeated loading and unloading of pipette tips; this

accumulation will affect the instrument’s performance. The RoboSep™ Tip Head Polishing Compound is specially formulated to help

remove the build-up of plastic residue and disinfect the tip head.

1. Apply 1 - 2 drops of RoboSep™ Tip Head Polishing Compound to a paper tissue or other laboratory wipe.

2. Wipe the RoboSep™ tip head.

3. Using a new piece of tissue or laboratory wipe, polish the tip head to make sure that all traces of the cleaner are removed.

5.4 CLEANING ROBOSEP™ The preferred method to clean RoboSep™ is to lightly spray 70% ethanol or 70% isopropanol on the instrument’s external surfaces,

and wipe it off with a soft, clean paper towel. RoboSep™ can also be wiped down with a dilute bleach solution (e.g. 1 in 10 dilution of

5% sodium hypochlorite solution), but should then be wiped clean with water afterwards.

For the touchscreen, it is recommended that the 70% isopropanol or ethanol be sprayed on a soft, clean paper towel prior to wiping

down. Excessive direct spray on the touchscreen may cause fluid to seep behind the external screen surface and into electrical

components. Do not allow excess fluid to pool at the margins of the touchscreen.

Note: RoboSep™ is resistant to spills; however, it is important to avoid spilling water inside the instrument through openings in the

work surface such as around the carousel bearing column and the optical sensor opening.

If accidental spilling of biological material occurs in the carousel area of the instrument and a more thorough cleaning is needed, the

following procedure should be used:

1. Unload and remove the carousel (see Figure 19 on page 11).

2. Disinfect the carousel and magnets with a dilute bleach solution and thoroughly rinse clean with water.

3. Rinse with distilled water.

4. Disinfect the spill with dilute bleach solution. Dispose of clean-up material according to local regulations.

5. Rinse the work surface tub with distilled water, then clean with 70% isopropanol or ethanol.





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5.5 TIP STRIP FAILURE DETECTION RoboSep™ has an automatic tip strip failure detector. If the unit detects a tip strip failure, a dialog box will open (Figure 37) and the

machine will beep. Pressing the “Resume” button will allow RoboSep™ to try stripping the tip again. Following a successful tip strip, a

second dialog box will open (Figure 38). If the tip strip is still not successful, the tip can be removed manually. If a tip strip failure

occurs, the tip head may need to be cleaned as described in Section 5.3.

5.6 REPLACING THE FUSES The two fuses are located at the bottom right side of the instrument, next to the power supply outlet and the power switch. If

RoboSep™ does not turn on when plugged in and the power switch is activated, the fuses may need to be replaced. To replace the

fuses:

1. Disconnect the power cable from the power source.

2. Disconnect the power cable from the instrument.

3. Open the fuse box and examine the fuses.

4. Replace the faulty fuse(s). Always use Type F 250V 4A 5X20mm fuses.

5. Reinsert the fuse box containing the new fuse(s) in its slot. Press gently until it snaps into place.

6. Reconnect the power cable to the instrument, then to a grounded (three-prong) power source.

Figure 37 – Dialog box shown if a tip strip failure is detected

Figure 38 – Dialog box shown following tip strip failure recovery





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6 TROUBLESHOOTING

RoboSep™ presents error messages to help diagnose some types of problems that may occur during use. Please record the message before calling Technical Support. The following tables address other potential issues that are not flagged as errors.

PROBLEM POTENTIAL CAUSE SOLUTION

RoboSep™ screen does not turn on. No sound, movement, or sign of activity within 30 seconds of attempting to power up the instrument.

Faulty fuse. Check that the fuses are correctly installed in described in Section 5.6.

No power to outlet. Check that the outlet is active by connecting a known working device such as a lamp.

Operating system boots up but RoboSep™ fails to fully initialize.

Power switch not left on long enough. It takes up to 10 seconds for the electronics to fully reset after the power switch has been turned off.

After shutdown, turn off the power switch and count to 10 before turning the power back on again.

RoboSep™ won’t resume a paused run. Lid up. RoboSep™ will not run if the lid is in the raised position.

Lower the lid and press Resume on screen.

RoboSep™ won’t shut down because the basic shutdown protocol won’t load.

Previous run not complete or needs to be unloaded.

Let run finish. Press unload button on the Sample Processing screen.

Reagents left in tips. RoboSep™ leaves a residual volume in tips when pipetting small volumes.

This is a normal operation and is required to meet the accuracy requirements for low volume reagent addition. The residual volume is 20 µL or less.

Missed tip pick-up.

Tip rack not placed properly on the carousel. Make sure that the tip rack is seated properly by pushing down firmly on the outside of the exposed portion.

Robotic arm or carousel was bumped during operation.

Only operate with the lid down.

If the above measures fail to solve the issue, contact Technical Support.

Tip head spits hydraulic fluid. Hydraulic fluid line contamination.

Fill hydraulic fluid bottle with 70% isopropanol or 70% ethanol and prime >4 times. Leave alcohol in line for >1 hour (longer times preferred), prime >4 times with sterile deionized water. If problem persists, contact Technical Support.

Damage to fluid line. Contact Technical Support.

Tips not well aligned with tubes. Positional calibration is required. Contact Technical Support.

Tip rack separates at the snap joint when removing cap.

Excess force applied at this joint by mistake. Ensure only the top cap is removed.

Missed tip stripping action. Tip stripping arm not moving.

Arm is out of position. Power down the instrument, open the lid, and pull the tip stripping arm out. Restart RoboSep™.

Tip stripping arm misaligned. Contact Technical Support.

Priming step missed or incomplete: No water ejected from the tip head prior to run.

Connections in hydraulic system are leaking air. Make sure the connection between the hydraulic fluid bottle and tubing is seated correctly by disconnecting and reconnecting firmly.

Hydraulic fluid bottle is empty. Refill bottle.

Can’t load protocol in Protocol Editor. File is read-only. Change file property.

Custom protocol does not appear in protocol database.

File label is not unique. Create unique label for protocol using Protocol Editor (see Section 3.3).





26

7 SPECIFICATIONS

Capacity - Labels and separates up to 4 samples simultaneously

- Up to 8 x 109 total cells (four samples of up to 2 x 109 cells each)

- Sample volume: from 250 µL to 8.5 mL for each sample

- Pipetting range: 12.5 µL to 5 mL (accuracy: volume variations <10% over whole pipetting range)

Dimensions and weight

- Height with lid: 56 cm (22 in)

- Width: 70.7 cm (27 in)

- Depth: 39.2 cm (15 in)

- Weight: 32.6 kg (72 lb)

Power Requirements - Electrical supply 100-120/220-240 V AC, 50/60 Hz, 260 W

- Uses two Type F 250V 4A 5X20mm

Conditions for Operation

- Temperature 10°C to 30°C

- Relative humidity 20 - 85% (non-condensing)

- RoboSep™ is not specified to be used in a cold room (4°C, 39°F)

- For indoor use only

Certifications - Pollution degree 2

- Installation category II

- Altitude 2000 m

- CSA certificate 1660865

- UL 61010-1

- CE marked

RoboSep™ is designed and manufactured so that it does not endanger the safety of human operators when properly

installed and maintained and used in applications for which it was intended. RoboSep™ meets the requirements of EC

directive 2006/95/EC (low-voltage directive) and 2004/108/EC. RoboSep™ meets the requirements of CAN/CSA-C22.2 No.

61010.1-04 and UL standard No. 61010-1 (safety requirements for electrical equipment for measurement, control and

laboratory use).

The RoboSep™ unit has a symbol affixed to the side, similar to the following:

This symbol denotes that the device must be disposed of in accordance with the Waste Electrical and Electronic Equipment

(WEEE) Directive.

RoboSep™ has been designed for easy dismantling and is in compliance with the recovery, reuse, and recycling targets set

out in the WEEE Directive.

RoboSep™ should not be disposed of at civic disposal sites.

Please contact STEMCELL Technologies for information about the meaning of the WEEE symbol and for information about

return and collection points for the device at the end of its life.

7.1 FACTORY CALIBRATION Dispensing, robotic positioning, and the touchscreen used on RoboSep™ are all factory calibrated. Adjustments to any settings should

be performed only by qualified personnel.

7.2 SECURITY Secured access to RoboSep™ can be implemented by connecting RoboSep™ to your institution’s network. These approaches prevent

unauthorized access to the RoboSep™ software, and may require IT support. Additional user profiles can also be set up on stand-

alone units, as RoboSep™ operates on a Windows XP platform. This is not the standard set-up; contact STEMCELL Technologies

Technical Support for additional information. A fee for secured instrument set-up may apply.

Copyright © 2013 by STEMCELL Technologies Inc. All rights reserved including graphics and images. STEMCELL Technologies & Design, STEMCELL Shield Design, Scientists Helping Scientists, EasySep, and RoboSep are trademarks of STEMCELL Technologies Inc.





27

8 ROBOSEP™ SPARE PARTS AND ACCESSORIES

PRODUCT NAME CATALOG #

RoboSep™ Service Rack 20101

Hydraulic Fluid Bottle and Tubing 20102

RoboSep™ Buffer (250 mL) 20104

5X RoboSep™ Buffer (250 mL) 20124

EasySep™ RBC Lysis Buffer 10X Concentrate (100 mL) 20120

RoboSep™ Filter Tip Racks (1 box of 8 racks) 20125

RoboSep™ Tip Head Polishing Compound (7 mL) 20119

Ethernet Cross-over Cable 20106

RoboSep™ Carousel 20107

“The Big Easy” EasySep™ Magnet 18001

RoboSep™ Barcode Scanner 20501

9 WARRANTY

The following is an overview of the warranty and service options available for RoboSep™. For answers to any maintenance or warranty

questions, please contact STEMCELL Technical Support (contact information is available in Section 10).

WARRANTY PLAN COVERAGE COVERAGE DETAILS CATALOG #

RoboSep™ Limited Warranty1 1 Year2

• Parts • Labor

20200

RoboSep™ Limited Warranty with Preventative Maintenance Package 1 Year2

• Parts • Labor • Preventive Maintenance3

20202

Preventative Maintenance Package 1 Year2 • Preventive Maintenance 20203

1 Included in purchase of RoboSep™. 2 Maximum purchase of up to 5 consecutive years. 3 Preventative Maintenance includes: Full calibration check, touchscreen verification, tip head verification, tip stripping verification,

hard-ware verification, soft-ware verification and/or upgrades, robot homing verification, fluidic system verification, electrical card and

connector verification, pump and drive verification, fastener and tubing verification, belt and motor verification, bearing and gear

verification, verification certificate, related shipping costs (if applicable).

Scientists Helping ScientistsTM | WWW.STEMCELL.COM

TOLL FREE PHONE 1 800 667 0322 • PHONE +1 604 877 0713

[email protected] • [email protected]

FOR GLOBAL CONTACT DETAILS VISIT OUR WEBSITE


DOCUMENT #28940 VERSION 2.4.0

STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485 MEDICAL DEVICE STANDARDS.

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